Method for treatment of cancer and infectious disease

ABSTRACT

In a method of treating a viral infection involving activation of NK cells, the improvement comprising administering to a patient receiving such treatment an effective NK-cell-activity facilitating amount of a histamine H 2  or 5-HT 1A  receptor agonist.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/465,432, filed on Dec. 21, 1999, now U.S. Pat. No. 6,155,266 which isa continuation of U.S. patent application Ser. No. 09/033,110, filedMar. 2, 1998, now U.S. Pat. No. 6,003,516 which is a continuation ofU.S. patent application Ser. No. 08/374,787, filed May 8, 1995, now U.S.Pat. No. 5,728,378.

FIELD OF THE INVENTION

The present invention concerns a pharmaceutical preparation or systemfor activation of natural killer cells (NK-cells), in order, forexample, to treat tumors or virus infections.

BACKGROUND OF THE INVENTION

Natural killer cells (NK-cells) are a group of spontaneously cytotoxiclymphocytes that destroy tumor cells by lysis with no antigenspecificity or restriction by histocompatibility molecules. Monocytesare involved in the regulation of the NK-cell's function, both throughmechanisms involving cell contact and through providing soluble NKcell-regulating mediators. Recently, a cell contact-mediated mechanismhas been described whereby monocytes regulate NK-cells. This type ofmonocyte-mediated regulation is exerted by monocytes that are obtaineddirectly from peripheral blood through counterflow centrifugalelutriation (CCE) and is regulated by the biogenic amines histamine andserotonin (Hellstrand and Hermodsson, 1986, J. Immunol. 137, 656-660;Hellstrand and Hermodsson, 1987, J. Immunol. 139, 869-875; Hellstrandand Hermodsson, 1990, Scand. J. Immunol. 31, 631-645; Hellstrand andHermodsson, 1990, Cell. Imnmunol. 127, 199-214; Hellstrand, Kjellson andHermodsson, 1991, Cell. Imrnunol., 138, 44-54). These NK-cell regulatingmechanisms caused by biogenic amines should be of importance to theNK-cell-mediated defense against metastatic tumors in vivo (Hellstrand,Asea and Hermodsson (1990), J. Immunology 145, 4365-4370).

Interferon-α(IFN-α) is an important regulating factor for NK cells. Iteffectively enhances the NK cell's cytotoxicity (NKCC) both in vivo andin vitro (Trinchieri, 1989, Adv. Immunol. 47,187-376; Einhorn, Blomgrenand Strander, 1978, Int. J. Cancer 22, 405-412; Friedman and Vogel,1984, Adv. Immunol., 34, 97-140).

Owing to the high rate of cancer and the only partially successfultreatment methods available today, there is a constant demand for otherimproved methods of treatment of tumors. There is also a great demandfor improved treatment methods for virus infections.

SUMMARY OF THE INVENTION

The goal of the invention is to create a pharmaceutical preparation orsystem that effectively stimulates NK cells, e.g., in order to treattumors, primarily myelomas, renal cancer, leukemias and melanoma, or totreat virus infections, primarily chronic hepatitis B and hepatitis C.The preparation or system according to the invention involves a firstcomposition, containing interferon-α or analogues thereof, and a secondcomposition containing at least one substance with histamine H₂, orserotonin 5-HT_(1A) receptor agonist activity, whereby said first andsecond compositions are either mixed in a preparation or supplied inseparate doses in an amount sufficient for the intended treatment. Theinvention also comprises a method for treatment of viral or neoplasticdisease comprising the step of coadministering interferon-α and aneffective amount of a histamine H₂ receptor agonist or a serotonin5-HT_(1A) receptor agonist. Furthermore, the invention includes a methodfor the treatment of viral infection comprising administering ahistamine H₂ receptor agonist or a 5-HT_(1A) receptor agonist.

The invention shall be described in greater detail below, makingreference to reported in vitro experiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows in graph form the synergistic NK cell activation againstcultured target cells produced by IFN-α and histamine or serotonin forvarious concentrations of IFN-α(0-100 U/ml).

FIG. 2 shows in graph form the synergistic NK cell activation producedagainst freshly recovered human leukemic cells by IFN-α and histaminefor various concentrations of IFN-α(0-100 U/ml).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention is based on the unexpected discovery that IFN-α and thebiogenic amines histamine and/or serotonin produce a synergisticactivation of NK cells.

The experiments reported hereafter show that eluted monocyteseffectively suppress the activation of NK cells induced by IFN-α.Furthermore, it is shown that histamine or serotonin, which act throughdefined bioaminergic receptors, remove the monocyte induced suppressionand thereby restore the ability of the NK cells to respond to IFN-α.

Analogues of histamine with H2-receptor agonist activity or othercompounds with H2-receptor agonist activity and analogues of serotoninwith 5-HT,_(1A)-receptor agonist activity or other compounds with5-HT_(1A)-receptor agonist activity that are suitable for use in thepresent invention are known within the art and shall not be describedmore closely here. For example, these analogues can have a chemicalstructure resembling that of histamine or serotonin, but modified byaddition of groups that do not negatively affect the H₂ or 5-HT_(1A)receptor activities. Known H₂-receptor agonists include histamine,dimaprit, clonidine, tolazoline, impromadine, 4-methylhistamine,betazole and histamine congener derivatives such as:

described as compounds 1, 6, and 9 in Khan et al., J. Immunol., Vol. 137pp. 308-315. Known serotonin 5-HT_(1A) receptor agonists include8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin), ALK-3(cis-8-hydroxy-1-methyl-2-(di-n-propylamino)tetralin), BMY 7378(8[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione),NAN 190 (1-(2-methoxyphenyl-4-[4-(2-phthalimmido)butyl]pierazine HBr),lisuride, d-LSD, flesoxinan, DHE (dihydroergotamine), MDL 72832(8-[4-91,4-benzodioxan-2-ylmethyl-amino)butyl]-8-azaspiro[4,5]decane-7,9-dione), 5-CT (5-carboxamidotryptamine), DP-5-CT(N,N-dipropyl-5-carboxamidotryptamine), ipsapirone, WB 4101(2-[[[2-(2,6-dimethoxyphenoxy)ethyl]amino]methyl]-1,4-benzodioxane),ergotamine, buspirone, metergoline, spiroxatrine, PAPP(1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl) piperazine), SDZ(−) 21009((4(3-terbutylamino-2-hydroxypropoxy)indol-2-carbonic-acid-isopropylester),and butotenine.

IFN-α and histamine/serotonin can be administered separately or in thesame preparation. The method of administration can be either local orsystemic injection or infusion. Other methods of administration can alsobe suitable.

The compounds can even be administered intraperitoneally or in anotherparenteral method. Solutions of the active compounds in the form of freeacids or pharmaceutically acceptable salts can be administered in waterwith or without a tenside such as hydroxypropylcellulose. Dispersionsmaking use of glycerol, liquid polyethyleneglycols, or mixtures thereofwith oils can be used. Antimicrobial compounds can also be added to thepreparation.

Injectable preparations may include sterile water-based solutions ordispersions and powders that can be dissolved or suspended in a sterilemedium prior to use. Carriers such as solvents or dispersantscontaining, e.g., water, ethanolpolyols, vegetable oils and the like canalso be added. Coatings such as lecithin and tensides can be used tomaintain suitable fluidity of the preparation. Isotonic substances suchas sugar or sodium chloride can also be added, as well as productsintended to retard absorption of the active ingredients, such asaluminum monostearate and gelatin. Sterile injectable solutions areprepared in the familiar way and filtered before storage and/oradministration. Sterile powders can be vacuum-dried or freeze-dried froma solution or suspension.

All substances added to the preparation must be pharmaceuticallyacceptable and essentially nontoxic in the quantities used. Thepreparation and formulations that produce a delayed release are alsopart of the invention.

The preparation is supplied in dosage units for a uniform dosage and tofacilitate administration. Each dosage unit contains a predeterminedquantity of active components to produce the desired therapeutic effect,along with the requisite quantity of pharmaceutical carriers.

IFN-α can be administered in a quantity of around 1000 to 300,000U/kg/day, preferably around 3000 to 100,000 U/kg/day and morepreferably, around 10,000 to 50,000 U/kg/day.

The compounds with H₂, and 5-HT_(1A) receptor agonist activity can beadministered in a of quantity of around 0.1 to 10 mg/day, preferablyaround 0.5 to 8 mg/day and more preferably, around 1 to 5 mg/day.However other quantities can be administered with IFN-α as decided bythe treating physician. For substances other than biogenic amines withcorresponding receptor activity, doses producing an equivalentpharmacological effect shall be used.

Although it is stated in the examples that the administration was givenin a single dose, it is obvious that the compounds can be distributedover longer periods of time for treatment of virus infections or tumors.

The daily dose can be administered as a single dose or it can be dividedinto several doses, should negative effects occur.

EXAMPLES In Vitro Studies of IFN-α and histamine/serotonin.

This example illustrates the effect of human recombinant IFN-α andhistamine/serotonin, separately and in combination, on the NK cellcytotoxicity (NKCC) for human mononuclear cells (MNC).

MNC were obtained from peripheral venous blood from healthy human blooddonors by Ficoll-Hypaque centrifuging, followed by Percolldensity-gradient fractionation (Timonen and Saksela, 1980, J. Immunol.Methods 36, 285-291; Hellstrand and Hermodsson, 1990, Scand. J. Immunol.31, 631-645).

In the respective Percoll fractions, the high-density MNC (Percollfractions 1-4) were small lymphocytes with low baseline cytotoxicityagainst K562 target cells. After removal of the monocytes, thelow-density fractions 6-10 displayed high NKCC, consistent with earlierstudies. (Timonen and Saksela, 1980, J. Immunol. Methods 36, 285-291).

The target cells used in these experiments were K562, an NK-cellsensitive erythroleukemic cell line, or Daudi, a relativelyNK-insensitive EBV-transformed B-cell lymphoblastoid cell line.

The NKCC was determined six times as the specific ⁵¹Cr-release for aMNC: target-cell ratio of between 30:1 and 3.8:1 in two-fold dilutiongradients. The suspensions of MNC/target cells were incubated inmicroplates at 37° C. for 6 hours (Daudi) or 16 hours (K562). Thesupernatant solution was then collected and examined for radioactivityin a gamma counter. The maximum ⁵¹Cr-release was measured in target cellcultures treated with Triton X-100. The NKCC was calculated as the celllysis % by the formula 100×(experimental release−spontaneousrelease/maximum release−spontaneous release)=cell lysis %.

A low-density Percoll fraction was separated by counterflow centrifugeelusion (CCE) in a monocyte and in a lymphocyte fraction. The monocytefraction was concentrated to >90% purity whereupon the contaminatingcells consisted of large lymphocytes. The lymphocyte fractions obtainedby CCE contained <3% monocytes, determined by morphology and Leu-M3(CD14) antigen expression. The lymphocytes were CD3⁻/16⁺/56⁺ T cells(45-50%), CD3⁻/16⁻/56⁻ NK cells (35-40%), CD3⁺/16⁻/56⁻ T cells (45-50%),CD3⁺/16⁺/56⁺ cells (1-5%), determined by flow cytometry.

The eluted monocytes and/or the NK cell-concentrated low-densitylymphocytes were treated with IFN-α and histamine/serotonin. Thecompounds were added, separately or in combination, to mixtures of MNCand K562 target cells at the start of a 16-hour ⁵¹Cr-release assay. Thecytotoxicity against K562 in the NK cell-concentrated lymphocytefraction was increased by IFN-α and unaffected by histamine orserotonin. The eluted monocyte fraction exhibited a low baselinecytotoxicity and was slightly induced by histamine/IFN-α orserotonin/IFN-α; this cytotoxicity resulted from the low fraction ofcontaminating lymphocytes (data not given). The addition of elutedmonocytes to the NK cell concentrated lymphocytes suppressed thebaseline cytotoxicity to K562. Furthermore, the eluted monocytes almosttotally inhibited the activation of the cytotoxicity by means of IFN-α(Table 1).

Histamine and serotonin restored the basal cytotoxicity of lymphocytesin mixtures of monocytes and lymphocytes. Furthermore, both histamineand serotonin eliminated the monocyte induced inhibition of the NK cellresponse to IFN-α. Hence, IFN-α plus histamine or serotoninsynergistically enhance the cytotoxicity in mixtures of monocytes and NKcell-enriched lymphocytes (Table 1).

In the experiments reported in Table 1, eluted lymphocytes were mixedwith monocytes as shown in the table, in a total volume of 150 μl. Thedata are NKCC (mean±SEM) of six determinations. Serotonin 10⁻⁴ M and/orIFN-α (25 U/ml) was added at the start of a 16-hour microcytotoxicitytest against 10⁴ K562 target cells.

TABLE 1 Suppression of NK Cell Cytotoxicity by Monocytes and Eliminationof This Effect with Serotonin NK CELL CYTOTOXICITY Mono- Lympho- AFTERTREATMENT WITH cytes cytes Serotonin + (× 10⁻⁴) (× 10⁻⁴) ControlSerotonin IFN IFN 0 12 34 ± 1 34 ± 3 58 ± 3 60 ± 2 6 12 10 ± 2 31 ± 2 17± 1 52 ± 2 12  12  9 ± 1 31 ± 2 10 ± 1 52 ± 2

Table 2 shows the synergistic activation of NK cells by combinedtreatment with IFN-α and histamine. Monocytes were recovered along withNK cells in low-density Percoll fractions. In the experiment shown inTable 2, IFN-α and/or histamine was added to MNC obtained from thesemonocyte-containing Percoll fractions. As was the case with mixtures ofeluted monocytes and low-density lymphocytes, IFN-α was relativelyineffective in these cell fractions, while histamine increased thecytotoxicity. Treatment of monocyte-containing cells with histamine(10⁻⁴-10⁻⁶ M) and IFN-α (25 U/ml) produced a synergistic NK-boostingresponse against K562 and against Daudi target cells. A similar resultwas obtained when histamine was replaced by serotonin.

In the results shown in Table 2, MNC from five different donors wereused. All pounds were added to mixtures of MNC and target cells at thestart of a 6 h (Daudi) or 16 h (K562) effecter and target cellincubation. The effecter cells were obtained from Percoll fractions 7-8,containing 33-55% monocytes.

FIG. 1 shows the synergistic NK cell activation by IFN-α andhistamine/serotonin for different concentrations of IFN-α (0-100 U/ml).Cells from the monocyte-containing Percoll fraction 8 were incubatedwith culture medium, histamine (10⁻⁴ M) or serotonin (10⁻⁴ M) in thepresence of IFN-α (0-100 U/ml). The data shown are NKCC (cell lysis %;mean ±SEM of six determinations). The compounds were added at the startof a 16 h microcytotoxicity test against K 562 target cells.

TABLE 2 Synergistic Activation of NK Cells by Histamine and IFN-α NKCC(cell lysis % ± SEM) Target MNC/target Histamine concentration Exp cellcell ratio Treatment 0 10⁻⁴ M 10⁻⁵ M 10⁻⁶ M 1 K 562 15:1 Medium 33.1 ±0.5  55.5 + 1 54.7 ± 1 39.2 ± 1 IFN 25 U/ml 33.1 ± 1   76.4 ± 3 74.1 ± 166.0 ± 2 2 K 562 15:1 Medium 20.7 ± 0.4  32.4 ± 1 27.4 ± 1 23.2 ± 2 IFN25 U/ml 27.4 ± 1   67.9 ± 2 66.2 ± 1 55.4 ± 1 3 K 562 15:1 Medium 31.4 ±1   43.3 ± 1 38.6 ± 1 29.4 ± 1 IFN 25 U/ml 32.5 ± 1   71.9 ± 1 66.5 ± 256.3 ± 2 4 Daudi 30:1 Medium 1.0 ± 0.4  4.4 ± 1  3.5 ± 2   1.1 ± 0.3 IFN25 U/ml 1.1 ± 0.5 31.7 ± 1 28.3 ± 1 14.1 ± 1 5 Daudi 30:1 Medium 2.2 ±1   13.5 ± 1  9.7 ± 1  2.5 ± 1 IFN 25 U/ml 2.7 ± 1   61.3 ± 3 52.3 ± 231.7 ± 1

The effect of histamine on monocyte-induced suppression of resting andIFN-α-activated NK cells was completely blocked by simultaneoustreatment with the specific H₂R antagonist ranitidine and imitated bythe H₂R agonist dimaprit, which is shown in Table 3. This means that theeffect of histamine on the NK cell's response to IFN-α is H₂R-specific.

TABLE 3 Effects of Histamine and H₂R Agonist Dimaprit and H₂-AntagonistRanitidine on NK Cells NKCC (Cell Lysis %) ± SEM AFTER TREATMENT WITHTreatment Control Ran IFN Ran + IFN Control 0.1 ± 0.1 0.0 ± 0.1 0.1 ±0.1 0.0 ± 0.1 Histamine 9.4 ± 0.3 1.5 ± 0.3 31.7 ± 0.3  1.6 ± 0.2Dimaprit 6.4 ± 1   0.4 ± 0.4 32.6 ± 1   0.5 ± 0.5

In the experiment shown in Table 3, culture medium (control), histamine(10⁻⁴ M), dimaprit (10⁻⁴ M), ranitidine (ran) (10⁻⁴ M) and/or IFN-α (25U/ml) were added at the start of a 6-hour ⁵¹Cr release assay using Dauditarget cells. The data are representative of three similar experiments.NKCC is given as mean cell lysis %±SEM of six determinations. Theeffecter cells were recovered from a low-density Percoll fraction 8,containing around 40% monocytes.

Serotonin acted synergistically with IFN-α and had an effectcorresponding to that of histamine. Ranitidine (10⁻⁴ M) did not alterthe effect of serotonin. The specific synthetic 5-HT_(1A) R-agonists8-OH-DPAT and (+)-ALK-3, which lack activity for 5-HT₁₈R; 5-HT₁₀R,5-HT₂R or—HT₃R, intensified the baseline NKCC and restored the NK cell'sresponse to IFN-α with a potency and effect comparable to that ofserotonin. This is shown in Table 4. Ketanserin and ondansetron, whichare antagonists of 5-HT₂R and 5-HT₃R, respectively, did not influencethe effect of serotonin in equimolar concentrations.

TABLE 4 The Effect of Serotonin and 5-HT_(1A)R Agonists on NK Cells NKCCAFTER TREATMENT WITH Treatment Medium IFN Medium 1.1 ± 1   0.5 ± 0.3Serotonin 10⁻⁴ M 10.4 ± 1   44.3 ± 1   Serotonin 10⁻⁵ M 4.5 ± 0.3 33.2 ±1   Serotonin 10⁻⁶ M 2.2 ± 0.4 12.3 ± 1   8-OH-DPAT 10⁻⁴ M 8.8 ± 1  43.3 ± 1   (+)-ALK-3 10⁻⁴ M 9.1 ± 1   40.4 ± 1  

In the experiment shown in Table 4, culture medium (control), scrotonin,8-OH-DPAT (+)-ALK and/or IFN-α (25 U/ml) were added at the start of a6-hour ⁵¹Cr-release assay against Daudi target cells. The NKCC is givenas cell lysis %±SEM of six determinations. The effector cells wererecovered from the low-density Percoll fraction 7, containing around 36%monocytes.

Similar experiments were then performed using freshly recovered humantumor cells as target cells, rather that the cultured tumor cell linesused as target cells in the experiments described above.

MNC were obtained from peripheral venous blood by Ficoll-Hypaquecentrifuging and the mononuclear cells were separated into monocytes andNK-cell-enriched lymphocytes (Hellstrand et al., J. Interferon Res., 12,199-206 1992). Seventy thousand NK-cell-enriched lymphocytes were mixedwith 70,000 monocytes and 20,000 ⁵¹Cr-labeled leukemic target cells (97%pure acute myelogenous leukemic cells) in a total volume of 150 μl. Thecells were treated with culture medium (control) or histaminedihydrochloride at a final concentration of 10⁻⁴ M, during a 16 hour⁵¹Cr-release assay to determine killed target cells.

The results are shown in FIG. 2. The data are the mean percent celllysis of six determinations ±SEM. The recorded cytotoxicity wascompletely depleted after removal of NK-cells using DYNABEADS coatedwith anti-CD56, but not by removal of T-cells using beads coated withanti-CD3 (Hellstrand et al., Scand. J. Immunol., 37:7-18 (1993). As seenin FIG. 2, treatment with interferon alone does not induce killing ofleukemic target cells unless histamine is present. In addition, it hasbeen shown that the cytotoxic effects obtained with histamine andinterferon-α are seen not only in cultured tumor cells, but in freshlyrecovered human leukemic cells as well.

Thus, in conclusion, it can be affirmed that the above-described invitro experiments demonstrate that the biogenic amines histamine,through H₂ type receptors, and serotonin, through 5-HT_(1A) typereceptors, abolish the monocyte-induced suppression of resting and IFN-αactivated NK cells. Treatment with IFN-α and compounds with H₂ orHT_(1A) receptor agonist activity thus produces a synergistic activationof NK cells, which can be used in connection with tumor treatment ortreatment of virus infections.

What is claimed is:
 1. In a method of treating a viral infection in apatient through activation of NK cells, the improvement comprisingadministering to the patient an effective NK-cell-activity facilitatingamount of a histamine H₂ or 5-HT_(1A) receptor agonist.
 2. The method ofclaim 1, wherein the receptor agonist is histamine.
 3. The method ofclaim 1, wherein the receptor agonist is a histamine congener.
 4. Themethod of claim 1, wherein the receptor agonist is serotonin.
 5. Themethod of claim 1, wherein the receptor agonist is selected from thegroup consisting of dimaprit, clonidine, tolazoline, impromadine,4-methylhistamine, betazole, 8-OH-DPAT(8-hydroxy-2-(di-n-propylamino)tetralin), ALK-3(cis-8-hydroxy-1-methyl-2-(di-n-propylamino)tetralin), BMY 7378(8[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione),NAN 190 (1-(2-methoxyphenyl-4-[4-(2-phthalimmido)butyl]pierazine HBr),lisuride, d-LSD, flesoxinan, DHE (dihydroergotamine), MDL 72832(8-[4-91,4-benzodioxan-2-ylmethyl-amino)butyl]-8-azaspiro[4,5]decane-7,9-dione), 5-CT (5-carboxamidotryptamine), DP-5-CT(N,N-dipropyl-5-carboxamidotryptamine), ipsapirone, WB 4101(2-[[[2-(2,6-dimethoxyphenoxy)ethyl]amino]methyl]-1,4-benzodioxane),ergotamine, buspirone, metergoline, spiroxatrine, PAPP(1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl) piperazine), SDZ(−) 21009((4(3-terbutylamino-2-hydroxypropoxy)indol-2-carbonic-acid-isopropylester),and butotenine.
 6. The method of claim 1, wherein said receptor agonistis administered in a daily dose of between about 0.1 and 10 mg.
 7. Themethod of claim 1, wherein said viral infection is a hepatitis virusinfection.
 8. The method of claim 7, wherein said hepatitis virus ishepatitis B.
 9. The method of claim 7, wherein said hepatitis virus ishepatitis C.
 10. A method for the treatment of hepatitis virusinfections in vivo, comprising: identifying a patient having a hepatitisvirus infection, and administering to said patient a first compositioncomprising interferon-α and a second composition having agonist activityfor histamine H₂ or 5-HT_(1A) receptors.
 11. The method of claim 10,wherein said first composition is administered in a daily dose of about1000 and 3000,000 U/kg.
 12. The method of claim 10, wherein said secondcomposition is administered in a daily dose of between about 0.1 and 10mg.
 13. The method of claim 10, wherein said first and secondcompositions are administered together.
 14. The method of claim 10,wherein said first and second compositions are administered separately.15. The method of claim 10, wherein said hepatitis virus is hepatitis B.16. The method of claim 10, wherein said hepatitis virus is hepatitis C.17. The method of claim 15, wherein the second composition compriseshistamine.
 18. The method of claim 16, wherein the second compositioncomprises histamine.